We have used the baculovirus expression system to produce a heavy meromyosin-like fragment that contains a truncated form of the 200 kD chicken nonmuscle myosin heavy chain-B (MHC-B). The truncated myosin heavy chain-B fragment of 1231 amino acids was coexpressed along with the 17 kD and the 20 kD myosin light chains. This was carried out by coinfecting intestinal insect cells with two viruses, one containing the truncated myosin heavy chain-B fragment and one containing both light chains. HMM-exp was purified from the insect cells following expression and found to consist of a 150 kD myosin heavy chain and two light chains in a 1:1:1 molar ratio. HMM-exp was soluble at low ionic strength, was bound to rabbit skeletal muscle actin in an ATP-dependent manner, was capable of moving actin filaments in an in vitro motility assay and manifested an actin-activated MgATPase activity provided that the 20 kD light chain was phosphorylated by myosin light chain kinase. Cloning of the cDNA encoding a chicken brain nonmuscle MHC-B revealed the presence of cassettes of amino acids near the ATP binding region and near the actin binding region which were located in the head region of the MHC (Takahashi et al., J. Biol. Chem. 267: 17864, 1992). In order to study the differences in the biological activity between the inserted and noninserted isoforms, we introduced the necessary nucleotides encoding the 10 amino acid insert near the ATP binding region into the cDNA encoding the myosin heavy chain-B in the baculovirus system. Following purification of both the expressed inserted and noninserted myosin heavy chain-B isoforms, we have been able to demonstrate that the former, but not the latter, was capable of serving as a substrate for proline- directed kinases in vitro. Tryptic phosphopeptide maps of the phosphorylated myosin heavy chain confirmed that the only site that was phosphorylated by these proline-directed kinases was in the inserted sequence of 10 amino acids present in the expressed myosin heavy chain-B isoform. We are presently characterizing the differences in biological activity between the inserted and the noninserted HMM-exp isoforms as well as the effect of phosphorylation on HMM-exp activity in vitro.